Identifying heterozygous base positions or small insertions or deletions in genomic DNA is often employed to locate mutations or polymorphisms in diploid organisms, detect genetic rearrangements, and uncover rare variants. The combination of the new Primer Designer™ Tool, BigDye® Direct Cycle Sequencing Kit and the Applied Biosystems® 3500 Series Genetic Analyzers for comprehensive, reliable sequencing data on targeted DNA regions and in heterogeneous mixtures, is critical for countless life science and research applications.

Step-by-Step Guide to Resequencing for Heterozygote Detection

DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction. Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction.

After the sequencing reaction, it is important to remove unincorporated dye terminators and salts that may compete for capillary electrophoretic injection. Unincorporated terminators can co-migrate with the sequencing template, resulting in basecalling.

Recommended Products: Purifying Sequencing Reaction

Which Sample Cleanup Product Is Right for You?

Cleanup Method

Description & Application

Product

Reagent-Based Sequencing Template Cleanup

Scavenges all unincorporated BigDye® terminators. Stabilizes samples before analysis. Best for long and short fragment recovery.

During capillary electrophoresis, the products of the PCR are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the fluorescent DNA fragments are separated by size and are detected by a laser/camera system.

After electrophoresis, Data Collection software creates a Sample File of the raw data. Using downstream software applications, further data analysis is required to translate the raw data into the corresponding electropherogram.